Discovery of CYP11A1 initiated metabolism of pro-vitamin D to 7? steroids (subject to UVB photoconversion to corresponding secosteroids) and sequential hydroxylation of vitamin D producing 20(OH)D3 and other hydroxyderivatives, defined new metabolic pathways of which the main intermediate, 20(OH)D3, is biologically active, while being nontoxic and noncalcemic in rats and mice at doses as high as 60 ?kg. These pathways can operate ex vivo in placenta, adrenal gland and epidermal keratinocytes. We also detected 20(OH)D3 in human serum. 20(OH)D3 is at least as potent as 1,25(OH)2D3 in anti-proliferative, pro-differentiation and anti-inflammatory assays and attenuates development of bleomycin induced fibrosis in mice. However, a major barrier for using 20(OH)D3 in preclinical models of scleroderma is a lack of understanding of the mechanism of its action on dermal fibroblasts. The goal of this R21 is to test hypothesis is that 20(OH)D3 acting directly on vitamin D receptor (VDR)- or/and on retinoic acid orphan receptor (ROR)- dependent mechanisms inhibit profibrotic fibroblast activities. To study this hypothesis one mechanistically oriented specific aim is designed with four subaims: 1. To test the mechanism of antifibrotic action of 20(OH)D3 in dermal fibroblasts. Subaim 1: We will investigate which signaling pathways utilized by TGF-?1 in dermal fibroblasts are inhibited by 20(OH)D3. We will determine whether this secosteroid inhibits other profibrotic effects of TGF-?1 and determine its relative potency on each pathway; Subaim 2: We will investigate the involvement of VDR-dependent pathways by testing the effects of 20(OH)D3 on fibroblasts derived from VDR-/- mice. Confirmations for humans will be carried out using dermal fibroblasts with receptors silenced by RNAi technology. These will be complemented by quantitative testing of ligand-induced VDR translocation to the nucleus and activation of VDRE transcriptional activity using VDR-GFP and VDRE-LUC constructs, respectively; Subaim 3: The hypothesis that 20(OH)D3 acting on ROR? and ROR? will regulate fibroblast activities will be tested. We will define interactions of 20(OH)D3 with ROR? and ROR? using biochemical and cell-based assays. Involvement of those receptors in the regulation of a phenotype will be evaluated using fibroblasts from ROR?- and ROR?-/- mice with further confirmation in human dermal fibroblasts with receptors silenced by RNAi. Divergence and overlaps between the actions on VDR, ROR? and ROR? will also be tested by whole genome RNAseq analysis supplemented by testing gene expression and bioinformatic analysis. This will define which phenotypic traits are regulated by VDR and which by ROR? or ROR?; Subaim 4: We will test whether antifibrotic activity of 20(OH)D3 is regulated by hydroxylation at C1? and/or C25. Techniques of biochemistry, gene silencing technology and cell biology will be used and will further be supplemented by pharmacological approaches. Defining which phenotypic treats are regulated through VDR or ROR? and ROR? by 20(OH)D3, would allow to perform future testing on proper KO mice to define role of the receptor in in vivo scleroderma models.